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Source: SARS-CoV-2 Research  Jun 09, 2021  8 days ago
BREAKING! SARS-CoV-2 Research: New Study By NIH Reveals That SARS-CoV-2 Nsp15’s Uridine Cleavage Can Evade The Immune Response!
BREAKING! SARS-CoV-2 Research: New Study By NIH Reveals That SARS-CoV-2 Nsp15’s Uridine Cleavage Can Evade The Immune Response!
Source: SARS-CoV-2 Research  Jun 09, 2021  8 days ago
SARS-CoV-2 Research: A new study by scientists from the US NIH has surprisingly revealed SARS-CoV-2 coronavirus utilizes a uridine-specific endoribonuclease, called nonstructural protein 15 (Nsp15), to evade the immune system. It works by cleaving viral RNA and preventing the activation of double-stranded RNA sensors. Although how Nsp15 recognizes its RNA target for cleavage remains unclear.

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Nsp15 is a uridine specific endoribonuclease that coronaviruses typically employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3’ of uridines, it is unknown if Nsp15 has any additional substrate preferences.
The study team used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization.
The tea also determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3’ of the cleaved uridine.
The study findings advance the understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.
The study findings were published on a preprint server and are currently being peer reviewed.
The study conducted by researchers from the National Institutes of Health in the USA shows that Nsp15 targets uridines (U). Following detection of U, Nsp15 can then further cleave a broad spectrum of RNA substrates.
Corresponding author, Dr Robin E. Stanley from the Signal Transduction Laboratory, National Institute of Environmental Health Sciences-National Institutes of Health, North Carolina told Thailand Medical News, “Overall, this work establishes SARS-CoV-2 Nsp15 as a largely non-specific endoribonuclease with recognition for a minimal consensus motif (N)(U)^(R>U>>C) (where N is any base and R is a purine). Our data show that Nsp15 acts in a distributive fashion to catalyze cleavage following uridines.”
The study team used a combination of techniques including cryo-EM, molecular dynamic simulations, and in vitro RNA cleavage assays to evaluate Nsp15’s substrate preferences beyond U. They performed a cryo-EM reconstruction of Nsp15 that’s RNA bound through information collected in its pre- and post-cleavage states.
The researchers looked at how the nucleotide 5’ and 3’ of uridine affected cleavage to determine RNA specificity. They also looked at how Nsp15 cleavages viral RNA substrates such as polyuridine and the transcriptional re gulatory sequence.
It was found that Nsp15 did not have any other base binding sites for RNA binding and recognition, indicating detection of U is critical for proper RNA cleavage.
Importantly it was discovered that several N-terminal domain residues from a nearby protomer interacted with the B2 adenine in the cryo-EM model. Considering they are essential for oligomerization and nuclease activity, the results suggest the N-terminal domain could help with engaging the RNA in the active site.
Hence, the structure-based point mutations suggest Nsp15 could be inhibited by disrupting the EndoU/NTD interface at the edge of the active site, which should destabilize the hexamer and lead to inactive monomeric enzyme,the study team stressed.
Interestingly N278 was the key residue found for maintaining Nsp15’s uridine preference. The residue is identical in Nsp15’s found in SARS-CoV-2 and MERS, but it differed across other coronaviruses. N278 interacts with S294 to move hydrogen bonding to favor uridines.
This was further confirmed with a variation of N278 showing reduced activity on cleaving uridine-containing substrates.
The study team noted that other factors not studied in this research cannot be ruled out and that there may be a possibility of others modulating sequence specificity and Nsp15’s preference towards uridine.
However, the study findings provide strong evidence towards the N278/S294 pair supporting a high preference for cleaving uridines.
Also substrate specificity points toward purines 3’ following a cleaved uridine
While Nsp15 can cleave polyuridine in RNA, there is a stronger preference between Nsp15 and the purines 3’ of the cleaved uridine outside of PolyU tracts. The results suggest Nsp15 evolved to have a strong preference for targeting uridine rather than cleaving randomly.
The study team concluded, “This work reveals that similar to RNase A, Nsp15 is a broad-spectrum endoribonuclease primarily guided to its cleavage targets by recognition of a single uridine.”
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